Biomarker dosimetry of acute low level of thermal neutrons and radiation adaptive response effect on rats

In this paper, we demonstrated the biological effects of acute low-dose neutrons on the whole body of rats and investigated the impact of that level of neutron dose to induce an in vivo radio-adaptive response. To understand the radio-adaptive response, the examined animals were exposed to acute neutron radiation doses of 5 and 10 mSv, followed by a 50 mSv challenge dose after 14 days. After irradiation, all groups receiving single and double doses were kept in cages for one day before sampling. The electron paramagnetic resonance (EPR) method was used to estimate the radiation-induced radicals in the blood, and some hematological parameters and lipid peroxidation (MDA) were determined. A comet assay was performed beside some of the antioxidant enzymes [catalase enzyme (CAT), superoxide dismutase (SOD), and glutathione (GSH)]. Seven groups of adult male rats were classified according to their dose of neutron exposure. Measurements of all studied markers are taken one week after harvesting, except for hematological markers, within 2 h. The results indicated lower production of antioxidant enzymes (CAT by 1.18–5.83%, SOD by 1.47–17.8%, and GSH by 11.3–82.1%). Additionally, there was an increase in red cell distribution width (RDW) (from 4.61 to 25.19%) and in comet assay parameters such as Tail Length, (from 6.16 to 10.81 µm), Tail Moment, (from 1.17 to 2.46 µm), and percentage of DNA in tail length (DNA%) (from 9.58 to 17.32%) in all groups exposed to acute doses of radiation ranging from 5 to 50 mSv, respectively. This emphasizes the ascending harmful effect with the increased acute thermal neutron doses. The values of the introduced factor of radio adaptive response for all markers under study reveal that the lower priming dose promotes a higher adaptation response and vice versa. Ultimately, the results indicate significant variations in DNA%, SOD enzyme levels, EPR intensity, total Hb concentration, and RDWs, suggesting their potential use as biomarkers for acute thermal neutron dosimetry. Further research is necessary to validate these measurements as biodosimetry for radiation exposure, including investigations involving the response impact of RAR with varied challenge doses and post-irradiation behavior.


Animal exposure to acute neutron radiation
After categorizing the animals into seven groups, Group #1 served as the sham control group.This group involved placing the rats in the same cages as those exposed to neutrons for same duration but without irradiation.Rats in groups #2 and #6 were exposed to acute neutron radiation with a total dose of 5 mSv of neutron radiation.After 14 days, group #6 was exposed to a single challenge dose of 50 mSv.For group #4, rats were exposed to only 50 mSv without exposure to neutron radiation.Then, 14 days later, rats were harvested for examination.In groups #3 and #7, rats were exposed to 10 mSv acute neutron radiation, while group #7 was exposed to a single challenge dose of 50 mSv.Whereas in group #5, rats were exposed to only 50 mSv, which was marked as a control group for RAR.After 24 h of the irradiation, rats were harvested for examination.In addition, the exposure of each group did not affect the other groups.Table 1 summarizes the plan for animal irradiation.

Hematological analysis
Blood samples were collected for hematological analysis in tubes containing EDTA (Anti Coagulate) by eye puncture via a capillary tube and were analyzed within two hours of collection 21 .The analysis of hemoglobin concentration (Hb), mean corpuscular volume (MCV), hematocrit (HCT), Red Cell Distribution Width (RDW), and mean corpuscular hemoglobin concentration (MCHC), a hematology Analyzer (Diff3) Mek6410/Mek-6420 was used, following Wintrobe's technique 22 .
Figure 1.Scheme of thermal neutrons irradiation and different planned parameters for measurements.The exposure was from an Am-Be source with an equivalent dose rate of 1.5 ± 0.06 mSv/h for all classified rat groups.

Lipid peroxidation analysis
The blood serum was used for lipid peroxidation analysis.In this test, blood samples were collected in plain tubes without anti-coagulant.The samples were then centrifuged for 10 min at 3000 rpm to separate the serum which was stored in a refrigerator at-20 °C for one week until used.To measure the lipid peroxidation product, malondialdehyde (MDA), a Thiobarbituric acid assay was performed.This assay is based on MDA's reaction with Thiobarbituric acid, resulting in Thiobarbituric acid reactive substances (TBARS), a red species that absorbs at 532 nm 23 .

Electron paramagnetic resonance (EPR) analysis
EPR measurements were performed using an X-band EPR spectrometer (Bruker EMX, Germany) at room temperature using a high-sensitive standard cylindrical resonator (ER4119HS) operating at 9.85 GHz, with a 100 kHz modulation frequency.The optimum EPR parameters were 2 mW microwave power, the modulation amplitude was 1 G, and the response time constant was 20 ms with a sweep time was 84 s.The optimum weight of dried blood powder was around 40 mg to fit 10 mm in height inside the tube.The EPR intensity, peak-to-peak height, of each lyophilized blood sample at room temperature by freezing and drying the resulting solution, was measured 3 times with 5 scans each run.The average values of the obtained measurements were normalized to weight and then plotted.Before all measurements, the standard sample of MgO doped with Mn 2+ was utilized independently to calibrate the EPR intensity, spectrometer stability, and the signal g-factor.

Biochemical examination
Similar to lipid peroxidation analysis, the serum was isolated in separate tubes without anti-coagulant materials for the biochemistry examination.It was performed by centrifuging the sera for 10 min at 3000 rpm and then storing it in a refrigerator at − 20 °C for almost one week before measurements.Catalase activity (CAT) was determined using Sinha 1972 approach by assessing the catalytic reduction of hydrogen peroxide 24 .The superoxide dismutase (SOD) activity was determined using Minami and Yoshikawa 1972 technique 25 .While Ahmed et al. 26 employed the method to measure the reduced glutathione (GSH) content.

Comet assay analysis
To detect the alkaline comet or single-cell gel electrophoresis (SCGE) assay, the technique reported by Singh et al. 27 was employed with slight changes to the measurement of comet assay (CA), tail length (TL), tail moment (TM), percentage of DNA in the tail (DNA%), and olive tail moment (OTMS).Due to the liver being an active organ in various biochemical processes in addition to the liver cells being sensitive to the DNA damage induced by radiation, the liver cells were chosen for the comet assay analysis.Following the animals sacrificing, 20 g of liver organs were collected and mixed with 10 ml of cold phosphate buffer solution (PBS) and centrifuged again for 15 min at 4000 rpm at 4 °C.The tissues were dried with sterilized absorbent paper.To prevent DNA damage, the liver cells were added to low-melting agarose.Approximately 1 × 10 4 cells were combined with low melting agarose (Solarbio, Beijing, China) and dispersed on microscope slides.The cells were lysed at pH 10, then the DNA was unwound for 20 min in an alkaline buffer (pH 13), followed by 30 min of electrophoresis (25 V, 300 mA).After neutralization (5 mg/L, Sigma, St. Louis, MO), the cells were stained with ethidium bromide.We utilized a fluorescence microscope (Nikon, Tokyo, Japan) loaded with CASP1.2.3 beta 1 software, developed by CaspLab-Comet Assay Software Project Lab which is freely available at: (https:// sourc eforge.net/ proje cts/ casp/) to assess all coded slides for each exposure condition.We measured the TL of 50 comets in micrometers, while TM was calculated from the comet tail length multiplied by the DNA percentage in the comet tail 28 .The data was decoded after completing all microscopic analyses.

RAR equivalent dose and RAR factor calculation
The term "RAR equivalent dose, RARED, (D RAR )" introduces a new concept indicating the actual equivalent dose with respect to changes in the investigated biological parameters due to the response of radio-adaptation.This definition allows for the measurement of animal ability to resist the effects of a larger challenge dose.It does so by comparing the extent of change in certain parameters when exposed to a high challenge dose after receiving small, acute priming doses with the change in these parameters after exposure to small, acute doses only.This  1) is derived in a similar manner to the supposed equation of Yonezawa effect scheme 29 , which is known as "the priming dose model", used for calculating the difference percentage due to radio-adaptation (δ) as in Eq. (2) 30 .
where (N p+c ) is the reading of the marker irradiated to priming and challenge doses and (N c ) is the reading of the challenge dose without the priming dose.At constant intervals between the priming and challenge doses and also between the challenge dose and the readout of markers, we proposed here an equation as a function of the equivalent doses only.RARED and RARF of acute doses of thermal neutrons to the radio-adaptive response doses proposed here were calculated from the calibration curve by fitting the measured marker data points.The RAR factor ( f RAR ) values range from 0 to 1.A value of zero indicates no RAR effect, while a value of 1 represents a full radio adaptive response (i.e.there is no scientific variation in the biomarkers between the zero dose and priming acute dose).This also means that the effect of RAR increases as the RARF increases from 0 to 1.

Statistical analysis
During our statistical analysis, we calculated the average results of five rats in each group, in addition to standard deviation (SD) values.Furthermore, we determined the degree of significance for p ≤ 0.05, where a difference is considered significant and meets the tolerance criteria.The values in the goodness of fitting column shown in all tables summarizing the fitting parameters of investigated biomarkers refer to p-values.Our approach involved the use of the statistical OriginPro 2021b program, developed by OriginLab (https:// www.origi nlab.com/), to fit all data.This included calibration functions and dose-response relationships for all parameters of acute neutron doses to radiation (5, 10, 50 mSv), and their reverse functions.

Declaration of animal treatment ethics
The authors declare that the animals were treated based on the guidelines of the National Institute of Health for human treatment of animals (USA) and the institutional animal care and use committee approved the work under approval number URAF-2-23.

Antioxidant enzymes for the blood
Figure 2 describes the effect of thermal neutrons on three antioxidant parameters: CAT, SOD, and GSH.As appeared in this figure, the exponential decay fitting of both GSH and CAT concentration for the groups that irradiated to 5, 10, and 50 mSv (G4) acute thermal neutron doses has a significant decrease in comparison to G1, ranging from 11.3 to 82.1% and from 1.18 to 5.83%, respectively, for G2 and G4.Also, there was a significant linear decrease (see Table 2) in the concentration of SOD enzyme for acute neutron doses when compared to the control group, as follows: 1.47, 6.09, and 17.8%.For RAR, a significant increase in all concentrations of antioxidant enzymes was observed for the 5 + 50 mSv groups (G6), where the values of GSH, CAT, and SOD increased by 200, 4.51, and 21.38%, respectively, in comparison to G5.While for G7, they have a slight and significant increase relative to G5, with a percentage of 73, 2.29, and 14.33%, respectively.Amongst the enzymes under investigation, the GSH enzyme has the highest response due to exposure to thermal neutron doses; moreover, it shows the best indication for RAR.

Lipid peroxidation in the blood
Figure 3a explains the effect of lipid peroxidation (MDA) on G1, G2, G3, and G4 as a result of exposure to acute doses of thermal neutron, while Fig. 3(i) depicts the effects of lipid peroxidation on all groups under investigation.As indicated in Fig. 3a, when compared to the control group, there is an elevation in the rate of the peroxidation process, but with a non-significant polynomial increase.Polynomial increase of lipid peroxidation in Fig. 3a for 5, 10, and 50 mSv (G4) neutron doses, with R = 0.9331, and the fitting parameters of this relation were summarized in Table 3.The MDA intensity of the G6 and G7 groups is less than that of G5, which was irradiated to a 50 mSv challenge dose (RAR), by a value of 60.34% and 29.38%, respectively.

Free radical intensity in the blood
The relationship between the concentration of radiation-induced free radicals, measured as EPR intensity, in the blood and acute doses of thermal neutrons, as well as the impact of the neutron dose on all groups, including the RAR effect, are portrayed in Figs.4a and i, respectively.EPR intensity for 5, 10, and 50 mSv (G4) neutron doses increased linearly, with R = 0.9996, and the fitting parameters of this relation were summarized in Table 4.The EPR intensity of the G6 and G7 groups is less than that of G5, which was irradiated to a 50 mSv challenge dose, by a value of 10% and 3.1%, respectively.This indicates obviously that the adaptation response after exposure to 50 mSv of thermal neutron occurred.

Blood hemoglobin and some blood parameters
The effect of various acute doses of thermal neutron on the concentration of some blood parameters (total Hb, HCT, MCHC, MCV, and RDWs) is presented in Fig. 6a-e for groups 1, 2, 3, and 4. Both Hb and RDWs, as shown in Fig. 6a and e have a significant change and were fitted linearly, where the slope of the regression line for Hb has a negative sign in contrast to that of RDWs, which has a positive value (see Table 6).The percentage of change in Hb concentration, relative to the control group, fell gradually till reached about 3.5% for 50 mSv, while for RDWs, it increased dramatically to 25.2% for the same dose.Whereas the MCV factor (Fig. 6d) has an exponential trend as a response to the neutron dose.On the other hand, HCT and MCHC indices have a fluctuated response, as noticed in Fig. 6b and c. Figure 6i-v depicts the responses of all groups, including the RAR groups study, demonstrating that the responses of both 5 + 50 mSv (G6) and 10 + 50 mSv (G7) groups, in all parameters except HCT and MCHC, have not followed the same trend of dose-response as the acute dose groups (G1-G4).The percentages of concentration change for MCV, and RDWs in G6 and G7 groups have a lower value when compared to G5 by (9.4 and 7.6)% and (16.4 and 14)%, respectively.Contrarily, total Hb has a higher concentration to some extent by a value of 2.3% for G6 and 0.3% for G7.This unambiguously indicates that all blood parameters under study, except HCT and MCHC, confirm the adaptation response behavior of rats exposed to 5 or 10 mSv prior to a relatively high dose of thermal neutrons.

RAR equivalent dose (RARED) and RAR factor (RARF)
The values of RARED were estimated from the calibration curve used for the fitting of measured marker data points and as a consequence, RARF values are calculated using Eq. ( 1) and summarized in Table 7.The mean values of these RAR equivalent dose for 5 + 50 mSv vary from 5.6 to 16.6 mSv for CAT and Hb parameters, respectively, with an average of 10.1 ± 3.4 mSv ( σ = 1 ), while the values for 10 + 50 mSv range from 16.5 to 40.9 mSv for the same parameters, respectively, with an average of 23.1 ± 7.5 mSv.The percentages of the standard variation between readings of G4 and G5 for all parameters studied in this paper are less than 4% , except OTM, DNA%, and MDA, which reach approximately 10% .On the other hand, the RARF values, as seen in Table 7, range from 0.79 for Hb to 0.99 for CAT, with an average of 0.91 ± 0.06 for 5 + 50 mSv.For the 10 + 50 mSv range, the values range from 0.49 for Hb to 0.89 for CAT, with an average of 0.78 ± 0.12.These results provide valuable insights for further analysis and interpretation.

Discussion
In the realm of radiation exposure, antioxidant enzymes are vital in protecting cells from the harmful effects of free radicals 31 , Reactive oxygen species (ROS) are removed by these enzymes to safeguard organisms from oxidative damage 32 .ROS is important for several biological processes, including cell differentiation, growth ) Dose (mSv)  www.nature.com/scientificreports/regulation, immunity, and programmed cell death 33,34 , and defense against microorganisms 35 .Ionizing radiation generates various types of ROS, such as superoxide, hydrogen peroxide, and hydroxyl radicals, which can cause severe damage to proteins, lipids, and nucleic acids.Furthermore, radiation damage to lipids is more severe due respectively, for the four acute dose groups (G1, G2, G3, and G4) that were exposed to acute neutron radiation for doses (0, 5, 10, 50 mSv).The impact of neutron dose on all groups as demonstrated in (i-v) for [CA], [TM], [DNA%], [TL], and [OTM], respectively.
to the lipid component in the membrane 36 .The body's defense mechanisms against oxidative stress caused by free radicals include preventive defenses, repair defenses, physical defenses, and antioxidant defenses.Enzymatic antioxidants, such as glutathione peroxidase (GSH), catalase, and superoxide dismutase (SOD), form a critical component of the antioxidant defense system.Under normal conditions, a balance between the intracellular levels and the activity of these antioxidants is maintained.The accumulation of free radicals due to an imbalance   The survival of organisms and their health depend on this equilibrium 37 .Antioxidants become oxidized and need to be replaced or renewed.Antioxidant enzymes, often found in cells, catalyze the destruction of several free radical species.Transition metal binding proteins inhibit the formation of extremely reactive hydroxyl radicals by blocking the interaction of transition metals such as iron and copper with hydrogen peroxide and superoxide, converting them into new products.Therefore, the decline in the concentrations of antioxidant enzymes can be attributed to their conversion into new products.As the equivalent dose of neutron increases, so do the concentrations of free radicals, which require more antioxidants to deactivate these radicals, resulting in a further drop in the concentration of antioxidants in the blood.There are numerous antioxidants, including GSH, CAT, SOD, and others 38 , which are effective scavengers of free radicals by giving electrons to ROS.They neutralize the negative effects of the latter, reducing oxidative stress and the oxidation of cell molecules 39 .The lack of antioxidant enzymes causes excessive oxidative stress, which increases the risk of disorder and negative treatment outcomes 40 .In response to an increase in free radicals, cells produce extra endogenous antioxidants such as catalase, glutathione, and superoxide dismutase that can reduce or eliminate damage to cell structure.SOD reduces superoxide ions to hydrogen peroxide, which is converted to water by catalase, while glutathione peroxidase lowers hydroxide ions 41 .
The effects of whole-body sub-lethal doses of gamma-ray exposure on plasma lipids and susceptibility to oxidative stress were studied in rats 42 .A significant elevation in MDA level and a reduced level in GSH content were found after radiation exposure indicating disorders of lipid metabolism.We studied the enzyme activities to confirm their contributions to the adaptive response.The results of the present study showed a low production of antioxidant enzymes that can be fitted exponentially in GSH and linearly in CAT and SOD in acute neutron exposure for both 5 and 10 mSv, then deep decreasing in the group of exposure to 50 mSv (G4) only.The exposure to 5 + 50 mSv (G6) of neutrons resulted in elevated concentrations of the antioxidant enzymes GSH, CAT, and SOD, accompanied by a decrease in free radicals when compared to 50 mSv (G5), which represented an adaptive response in rat blood cells.The RARED were 9.7, 5.6, and 7.4 mSv and RARF were 0.91, 0.99, 0.96 for GSH, CAT, and SOD, respectively.
Lipid peroxidation is a natural and necessary process that occurs in all cells and tissues at low levels, producing MDA and other byproducts 43 .Measuring MDA levels helps determine the impact of ROS on biological systems.Lipid peroxidation, which is caused by free radicals, is a process that oxidizes polyunsaturated fatty acids.Elevated lipid peroxides can interface with the biochemical and physiological processes of red blood cells 44 .Researchers investigated the radioprotective effect of recombinant human antioxidant enzymes on mice subjected to 5-7 Gy γ-irradiation.The study showed that rhCuZn-SOD had a significant radioprotective effect by eliminating free radicals, increasing the activities of SOD, GSH, and CAT in blood and liver cells, reducing MDA levels, enhancing the immune system response, and prolonging survival.Excess free radicals can lead to cell death, and exposure to gamma radiation can increase MDA levels 45 .The deleterious effects of excess free radicals, or oxidative stress, have been reported to eventually lead to cell death.Benderitter et al. 46 noted an increase in malondialdehyde MDA a few hours after gamma radiation exposure.Lipid peroxidation results showed an increase as the acute neutron dose increased, which is expressed using a quadratic polynomial function.Also, it showed a deep decrease in the lipid peroxidation process in the group of 5 + 50 mSv and a slight decrease in the 10 + 50 mSv group when compared to a challenge dose of 50 mSv (G5) only, indicating the occurrence of an adaptive response.
EPR is the only non-destructive method that directly detects paramagnetic species, such as free radicals 47 .This makes it an effective tool for determining the concentration of unpaired electrons in a sample, even if the specific free radical is unknown.Additionally, EPR can identify biological molecules that contain free radicals or transition metal ions (Fe 3+ , Cu 2+ , Mn 2+ , and Co 2+ ) 48 .Neutron, gamma, or X-ray radiation exposure stimulates the body to create free radicals.The results of our study indicate a slight increase in the production of free radicals in all acute exposure groups (G2, G3, and G4) for doses (5, 10, and 50 mSv).Although exposure to a neutron dose of 5 mSv caused higher free radicals, it showed a higher radio adaptive response through lower free radical density at exposure to 50 mSv after 5 mSv compared to exposure to a neutron radiation dose of 10 + 50 mSv.
The comet assay (CA) is a versatile, uncomplicated, and adaptable technique for evaluating DNA damage and repair at the cellular level.The CA enables the detection of early or acute DNA damage after brief exposure, which may be repaired or subjected to programmed cell death (apoptosis) and/or mutations, leading to less detectable DNA damage 49 .Radiation-related cell damage is also known to involve the formation of free radicals, including hydroxyl radicals, lipid peroxide radicals, superoxide radicals, and lipid radicals.Lipid peroxide radicals cause lipid peroxidation in biological membranes, resulting in various biological damages, along with direct DNA damage 50 .The hydroxyl radical reacts with all components of the DNA molecule, causing damage to both the purine and pyrimidine bases and the deoxyribose backbone 51 .Kumaravel and Jha 52 used a comet assay to detect the measure(s) most strongly linked to DNA damage following exposure to various levels of gamma radiation ranging from 1 to 8 Gy.The study recorded various parameters, including OTM, TL, TM, DNA%, and others.Despite the increasing trend of these parameters, the results in that study are not comparable to those of our current study because of the variation in the range of gamma dose.The comet test revealed less DNA damage in the cells of mice exposed to pre-irradiated gamma rays than in the cells of rats that only received the challenge dose 53,54 .
Adaptive Response is becoming increasingly important in biodosimetry or accidental risk assessment for occupational workers and radiotherapeutic patients, which are assayed on CA mutations as endpoints.The present study showed an increase in all comet assay parameters (TM, DNA%, TL, and OTM) after exposure to acute doses of neutron radiation.Low-dose irradiated groups (G2 and G3) showed lower DNA damage when compared to the irradiated groups with a challenge dose of 50 mSv (G6 and G7).The adaptive response was demonstrated upon exposure to neutron radiation for 5 mSv before the challenge dose of 50 mSv, yet it is less obvious after neutron exposure for 10 mSv before the 50 mSv challenge dose.The adaptive response was undoubtedly determined after receiving 5 mSv of neutron radiation prior to the 50 mSv challenge dose.In contrast, it was less notable after receiving 10 mSv of neutron radiation prior to the 50 mSv challenge dose, which is consistent with the findings of Gajendiran et al. 11 .The adaptive response seen in the comet assay findings indicates the evidence of a resistance to the induction of DNA alterations following the irradiation of rats that were previously exposed to acute low doses of neutrons below the annual permissible doses with low dose rates.
The total Hb concentration after acute neutron doses showed a significant change, which differs from the insignificant change observed in gamma doses reported by Attia et al. 55 .This difference could be due to various factors such as differences in irradiation beam quality, type of radiation, and the range of dose investigated.Additionally, the change in the total Hb concentration was slight and further complicated by age differences in the studied animals.RDWs provided a more sensitive measure of small variations in red blood cell size than peripheral smears for detecting mild and moderate degrees of Iron Deficiency Anaemia (IDA).As such, RDWs could be used as a successful diagnostic technique for IDA RDWs can also express small variations in different populations of red cell size 56 and correlates with the variation in red blood cell size (homogeneity or heterogeneity) equivalent to anisocytosis 57 .Thus, changes in the electrical properties of the cell membrane caused by oxidative damage by different free radicals could be responsible for the negative effects of radiation exposure on red blood cell shape and size distribution 58 .The study found increased values of MCV and RDWs in neutron exposure for 5, 10, and 50 mSv, indicating the presence of cells of widely differing sizes that might be a result of changes in the erythrocyte cell membrane.Oxygen-free radical species produced from exposure to ionizing radiation induce deleterious damage to the cell membrane.The group exposure to 5 mSv before exposure to dose (50 mSv) showed an adaptive response higher than the group exposure to 10 before 50 mSv irradiation as it reduced the membrane damage of 50 mSv exposure as shown in low MCV and RDWs results.
It is worth mentioning that RAR behavior can be noticed clearly in the G6 and G7 groups when compared with the RAR-control group (G5) in all studied parameters here except MCHC and HCT%.The enhancement of antioxidant parameters such as GSH, CAT, and SOD enzymes led to scavengers of free radicals, low production of lipid peroxidation, damage to DNA, and the volume and distribution width of red blood cells.This is an adaptation against thermal neutrons due to the exposure to priming doses of 5 and 10 mSv before the challenge dose of 50 mSv when compared to 50 mSv only (G5).The analysis of RAR factor values for all dosimetric markers under the study indicates that a lower priming dose leads to a higher adaptation response, while a higher priming dose results in the opposite effect.This suggests a dependence of the factor on the priming dose.
Ultimately, the fitting of the results reveals that the SOD, EPR, DNA%, RDWs, and Hb concentration parameters have a linear regression, while TL has a polynomial fitting.On the other hand, GSH, CAT, CA%, TM, OTM, and MCV responses to thermal neutrons can be exponentially fitted.All linearly fitted parameters with an R-value greater than 0.99 are the best choice for the bio-dosimeters.However, they do not have the same precision; DNA% is the most precise biomarker, whereas EPR has the greatest standard deviation.Further studies are still required for a complete understanding of the adaptive response and the dosimetry for low doses of thermal neutrons, such as the duration of the intervals between irradiations, long-term stability, and storage temperature effect.

Conclusion
Prolonged exposure to low levels of neutron radiation can result in elevated DNA damage, lipid peroxidation (MDA), free radicals, and mean corpuscle volume (MCV), as well as a decrease in antioxidants such as GSH, SOD, and CAT.The current research has identified DNA%, SOD enzymes, EPR intensity, total Hb concentration, and RDWs as biomarkers for assessing thermal acute neutron dose, with DNA% being the most effective biomarker for acute exposure.However, these suggested biomarkers need further investigations to confirm their reliability and applicability in the field of biodosimetry of thermal neutrons.The study also found that stimulation of the immune system occurred in the group exposed to 5 or 10 mSv prior to a 50 mSv challenge dose from thermal neutron, indicating that the rat was healed from the neutron radiation effect rapidly within two weeks.This has been proven by elevated concentrations of the antioxidants, a lack of free radicals, low lipid peroxidation, a reduction in the average volume of red blood cells (MCV), a narrowing of the red cell size distribution (RDWs), and a decrease in DNA damage, percentage of DNA in tail, and tail moment.
The radiation adaptive response behavior for the group (G6) exposed to 5 mSv prior to receiving a dose of 50 mSv is more obvious than it is for the group (G7) exposed to 10 mSv beforehand.It is worth noting that the lowest RARED, which was calculated as an acute neutron dose, is for catalase enzyme (CAT) at either 55 or 60 mSv, (5.6, 16.5 mSv) respectively, while the highest dose was for total Hb concentration (16.6, 40.9 mSv) respectively.The estimated average of RARED for both 55 and 60 mSv accumulated doses was about one-sixth and one-third of their actual doses, respectively.The calculated RAR factor indicates that the lower priming dose promotes a higher adaptation response and vice versa.The impact results of acute doses of thermal neutrons prompt us to investigate the effect of mixed fields of gamma and neutrons on biological systems.Despite this positive result of the radio-adaptive response, it is important to emphasize that acute doses of a neutron source still have harmful effects on some biological systems.Therefore, we strongly recommend carefully managing radioactive materials to prevent the harmful effects of ionizing radiation exposure, in line with the recommendations of international radiation organizations and committees.

Figure 5
Figure5demonstrates the dose-response of comet assay percentage (CA%), tail moment (TM), percentage of DNA in the tail (DNA%), tail length (TL), and olive tail moment (OTM) for the irradiated groups.Table5

Figure 3 .
Figure 3. (a) Shows the effect of acute neutron doses (0, 5, 10, and 50 mSv) on MDA concentration for the four groups (G1,2,3,4).The polynomial fit of relationship between the lipid peroxidation (MDA) and the acute neutron radiation dose was performed.(i) shows overall behavior of lipid peroxidation process for all groups.Error bars represent the standard deviation of five aliquots.

Figure 4 .
Figure 4. (a) The relation between EPR intensity for the four groups (G1, G2, G3, and G4) and the acute neutron equivalent dose.(i) shows overall changes on EPR intensity for all groups, indicating the adaptation response.

Table 1 .
Irradiation plan to different thermal neutron doses for all classified groups.*The equivalent dose rate of thermal neutrons is 1.5 mSv/h.

Table 2 .
The fitting parameters of the relation between antioxidant enzymes and neutron dose for the groups that were exposed to an acute dose of neutron radiation in addition to the control (zero dose) group.*Tolerance criterion satisfied.

Table 3 .
The fitting parameters of the relation between dose and Lipid Peroxidation for the groups that were exposed to acute doses of neutron radiation in addition to the control (zero doses) group.

Table 4 .
The fitting parameters of the relation between EPR intensity and dose for the groups that were exposed to an acute dose of neutron radiation in addition to the control (zero dose) group.

Table 7 .
The mean of the equivalent dose of RAR and the calculated factor of RAR for each marker under the study as an acute neutron dose for 5 + 50 and 10 + 50 mSv doses.Average and standard deviation values are in bold.